1. Field of the Invention
This invention relates to alpha-amidating enzymes, the production of alpha-amidating enzymes and their use in the production of alpha-amidated products by action of the enzymes on glycine-extended substrates. In certain preferred embodiments, the alpha-amidating enzyme of the invention may be used in the production of useful alpha-amidated hormones and products for agricultural or medical use including calcitonins, growth hormone releasing factors, calcitonin gene-related peptides, and other alpha-amidated products.
2. Prior Art
The intracellular processing (cleavage and/or functional group modification) of precursor forms of native proteins following their translation from nucleic acid coding sequences has been clearly documented.
In general, mammalian cells and other eukaryotes can perform certain post-translational processing procedures, while prokaryotes can not. Certain prokaryotes, such as E. coli, are widely employed as hosts for the production of mammalian proteins via recombinant DNA (rDNA) technology because they can be readily grown in batch fermentation procedures and because they are genetically well-characterized. However, many mammalian proteins produced by genetic engineering technology require some type of post-translational processing, and this must often be accomplished by using complex, in vitro chemical procedures which are cost-prohibitive for large-scale production applications.
One type of processing activity involves the specific amidation of the carboxyl-terminal amino acid of a protein. Many naturally-occurring hormones and peptides contain such a modification, which is often essential if the protein is to be biologically active. An example is calcitonin, where the substitution of a non-amidated proline residue for the amidated proline of the native form results in a 3,000-fold reduction in biological activity.
An agent which effects this C-terminal (alpha) amidation recognizes a glycine residue which immediately follows the amino acid to be amidated (R-X-gly, where R is the main body of the protein, X is the residue which is amidated, and "gly" is the glycine residue). The glycine is cleaved and actually donates the amino moiety to the penultimate amino acid, thereby amidating it.
The first authors to report an approximate molecular weight for an alpha-amidating enzyme were Bradbury A. F., et al., Nature, Vol. 298, 1982, p. 686-88. Using Sephadex G-100 they suggested a minimum apparent molecular mass of approximately 60,000 daltons.
Subsequent studies have suggested the molecular mass of such an enzyme to be between 60,000 and 70,000 daltons (as measured by gel filtration chromatography). These include Husain, I., and Tate S. S., FEBS Letters, Vol. 152 #2, 1983, p. 277-281; Eipper, et al., PNAS Vol. 80, 1983 p. 5144-5148; Gomez et al., FEBS Letters, Vol. 167, #1, 1984, p. 160-164, and Kizer, J. S., et al., PNAS, Vol. 81, 1984, p. 3228-3232.
Eipper et al., PNAS, Vol. 80, 1983, p. 5144-48, have reported that in addition to molecular oxygen, two co-factors are required for maximal enzyme amidation activity; these are ascorbic acid and copper (II) ion.
The chemical reaction resulting in the amidation of the carboxyl-terminus of a peptide requires a source for the amino group. Bradbury, A. F., et al., Nature, Vol. 298, 1982, p. 686-688, demonstrated that glycine is cleaved and donates the amino moiety to the penultimate amino acid, resulting in the amidation of the latter. The requirement for glycine as the amino group donor has been substantiated by other authors.
Landymore, A. E. N., et al., BBRC Vol. 117, #1, 1983, p. 289-293 demonstrated that D-alanine could also serve as an amino donor in the amidation reaction. Subsequent work by Kizer et al., PNAS, Vol. 81, 1984, p. 3228-3232, showed two distinct enzyme activities in rat brain which were capable of catalyzing the alpha-amidating reaction. The higher molecular mass species (70,000 daltons) has a specificity restricted for glycine at the carboxyl-terminus of the substrate. The lower molecular mass enzyme accepts a substrate with .beta.-alanine as the carboxyl-terminal amino acid.
The pH optimum for the alpha-amidating enzyme extracted and partially purified from porcine pituitary was reported by Bradbury A. F., and Smythe D. G., BBRC, Vol. 112, #2, 1983, p. 372-377 to be approximately 7.0. Eipper, B. A., et al., PNAS, Vol. 80, 1983, p. 5144-5148, corroborated these results by reporting a pH optimum of 7 for an alpha-amidating enzyme which was partially purified from rat pituitaries. They also noted that enzyme activity declined rapidly at pH levels below 6.5 or above 7.5.
In all of the aforementioned publications, (incorporated herein by reference), the extracts and partially purified enzyme mixtures described contain additional proteolytic enzymes capable of degrading potential substrates and products as well as alpha-amidating enzymes themselves, thus retarding the amidation by such enzymes of peptides and polypeptides purified from natural sources or produced by recombinant DNA techniques.
Broadly, all amidation activities previously measured by others were based upon the conversion of D-substrates such as a tripeptide, D-Tyr-Val-Gly-COOH to D-Tyr-Val-CONH.sub.2. Of the two possible configurations ("D" or "L"), naturally-occurring, biologically important amino acids occur in the "L" form. However, use of the "D" form by these other investigators was necessitated to counteract the presence of extraneous proteolytic enzymes in the impure amidating enzyme preparations used by these researchers. These extraneous enzymes may have a pronounced proteolytic effect on L-amino acid substrate while having little effect on a D-substrate. The investigators, saddled with proteolytic and other impurities in their enzyme, used unnatural "D" substrate in order to avoid some of the effects of the impurities. No one prior hereto has been able to demonstrate that their enzyme preparations can efficiently amidate any physiologically relevant substrates, i.e., L-substrates for conversion to biologically active alpha-amidated L-products.
As demonstrated herein, the preparations of this invention are capable of effectively amidating L-substrates and, on D-substrates, have an activity of from 60 to more than 1,000 times greater than the highest activity noted in any Prior Art of which applicants are aware.
Enzymatic preparations capable of amidating the carboxyl-terminus of peptides and proteins have been described from a variety of sources. For instance, Bradbury, A. F., et al., Nature Vol. 298, 1982, p. 686-688 (the entire disclosure of which is incorporated herein by reference) reports an alpha-amidating enzyme activity to be present in porcine pituitary. The preparation of porcine pituitary containing the enzyme has the ability to convert peptides that terminate in a glycine to the corresponding desglycine peptide amide. Bradbury et al. acknowledges, however, that the preparations will not amidate peptides or polypeptides purified from natural sources:
"An assay system for detecting and estimating amidating activity in pituitary was obtained by examining the ability of enzyme preparations to convert the synthetic tripeptide D-tyrosylvalylglycine to the corresponding dipeptide amide D-tyrosylvaline amide . . . The D-tyrosine residue conferred stability against degradation by aminopeptidases present in tissue homogenates . . . Control experiments showed that when synthetic . . . D-tyrosylvaline amide was incubated in the same conditions it was slowly degraded. Thus the formation of the dipeptide amide by the pituitary enzyme is followed by its destruction by other enzymes present in the pituitary extract." (page 686)
Thus, Bradbury et al. acknowledges that the preparations described contain other proteolytic enzymes which degrade the peptide or polypeptide and that the non-naturally occurring D-tyrosine residue was utilized to minimize such degradation.
Further, Bradbury et al. teaches the use of homogenization or sub-cellular fractionation followed by gel filtration chromatography to purify an amidating enzyme that, admittedly, remains contaminated with proteolytic enzymes.
Husain, I., and Tate, S. S., FEBS Letters, Vol. 152, #2, 1983, p. 277-281, describe an alpha-amidating activity present in bovine pituitary neurosecretory granules.
Eipper et al., PNAS Vol. 80, 1983, p. 5144-5148, reported an alpha-amidating enzyme activity to be present in the anterior, intermediate and posterior lobes of the rat pituitary gland and bovine intermediate pituitary. However, this reference only teaches the utilization of synthetic D-Tyr-Val-Gly substrate to search for alpha-amidation activity, a recognition of the impurity of the preparations produced.
Gomez et al., FEBS Letters, Vol. 167, #1, 1984, p. 160-164 determined that rat hypothalamus also contained an alpha-amidating enzyme activity.
Bradbury, A. F., Smythe, D. G., in Peptides Structure and Function: Proceedings of the Eighth American Peptide Symposium; p. 249-52 (1983), Editors Hruby, V. J., and Rich, D. H., describe the presence of an alpha-amidating enzyme activity in rat thyroid glands.
Mains R. E. et al., Endocrinology, Vol. 114, 1984, p. 1522-1530, reported that a murine cell line derived from the anterior pituitary lobe (ATT-20) contained an alpha-amidating enzyme activity that apparently decreased with time in culture.
Glands or organs known to contain amidated peptides may contain an enzyme capable of catalyzing the amidation reaction. For example, lower life forms such as the dog fish (Squalus acanthias) have been reported by O'Donohue T. L., et al., Peptides 3, 1982, p. 353-395, to contain amidated peptides in pituitary extracts. Scheller, R. H. et al., Cell, Vol. 32, 1983, p. 7-22 reported the presence of amidation signal peptides in the marine snail Aplysia. Despite the apparent ubiquitous distribution of this activity in nature, little information has been published on the physicochemical characteristics of the enzyme. This may be attributed to the very low levels of enzyme present in these neuroendocrine organs.
The presence of amidated peptides in a particular tissue is not necessarily synonymous with high levels of alpha-amidating enzyme. For example, rat anterior pituitary tissue contains high alpha-amidating activity but no known substrates [Eipper et al., PNAS 80, 5144-5148 (1983)]. Rat posterior pituitary tissue contains amidated peptides (oxytocin and vasopressin) but has very little alpha-amidating activity [Eipper et al., Endo 116, 2497-2504 (1985)]. Therefore, until individual tissues are tested for alpha-amidating activity, the presence or potential levels of the enzyme can not be anticipated.